X-ray microscope system with cryogenic handling system and method

ABSTRACT

A cartridge-based cryogenic imaging system includes a sample handling system. This system uses a kinematic base and cold interface system that provides vertical loading to horizontally mounted high-precision rotation stages that are able to facilitate automated high-resolution three-dimensional (3D) imaging with computed tomography (CT). Flexible metal braids are used to provide cooling and also allow a large range of rotation. A robotic sample transfer and loading system provides further automation by allowing a number of samples to be loaded and automatically sequentially placed on the sample stage and imaged. These characteristics provide the capability of high-throughput and highly automated cryogenic x-ray microscopy and computed tomography.

RELATED APPLICATIONS

This application claims the benefit under 35 USC 119(e) of U.S.Provisional Application No. 61/096,502, filed on Sep. 12, 2008, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Microscopy has played an important role in science and technology. Onearea where light and electron microscopy techniques have beenindispensable is biological sciences. Light microscopy has allowedobservation at 200 nanometer (nm) scale resolution, while electronmicroscopy has demonstrated atomic scale resolution with thin-sectionedspecimens. Recent developments in x-ray microscopy have allowed thickhydrated samples with tens of nanometer resolution.

For most effective observations, cells and biological tissues must beimaged in a hydrated state in order to have the highest fidelityrepresentation of the living state. But when imaging hydrated organicspecimens using ionizing radiation, radiation damage often limits thequality and resolution of the images that can be obtained. The solutionis to work with hydrated specimens that have been rapidly frozen so asto minimize the formation of crystalline ice in the specimens.

Cryogenic specimen handling methods were first developed in electronmicroscopy in 1974 by K. Taylor and R. Glaeser, see Electron diffractionof frozen, hydrated protein crystals. Science, 106:1036-1037, 1974, andby the late 1980s there was a considerable knowledge base in placeregarding rapid freezing and cryo electron microscopy. Cryomicroscopy isalso expected to be important in trace element mapping in fluorescencemicroprobes, since specimen drying is likely to affect the distributionof the diffusible ions that can play such important physiological roles.Cryogenic methods have also found wide spread use in proteincrystallography, where the usual practice involves a cryogenic gasstream directed onto a specimen to cool it within an atmosphericpressure, room temperature environment.

SUMMARY OF THE INVENTION

Aspects of the present invention concern cryogenic sample handlingsystems for high-resolution microscopy applications, such as x-ray,optical, and/or electron microscopy. By using a cartridge sample mountand robotic sample handling system, highly automated sample transfer andloading can be achieved. These are essential components of ahigh-throughput automated cryogenic microscopy that maintains thetemperature of the specimen at between 80 and 170 degrees Kelvin, forexample, or lower.

This system uses a kinematic mount and cold interface system thatprovide vertical loading to horizontally mounted high-precision rotationstages that are able to facilitate automated high-resolutionthree-dimensional (3D) imaging with computed tomography (CT). Flexiblemetal braids are used to provide cooling and also allow a large range ofrotation. A robotic sample transfer and loading system provides furtherautomation by allowing a number of samples to be loaded andautomatically sequentially placed on the sample stage and imaged. Thesecharacteristics provide the capability of high-throughput and highlyautomated cryogenic x-ray microscopy and computed tomography.

In general, according to one aspect, the invention features a cryogenicimaging system, comprising a kinematic base that receives cartridges ona cryogenic base, with each cartridge carrying a specimen. The systemfurther includes a positioning stage and a warm-cold interface betweenthe positioning stage and the cryogenic base. A flexible thermal linkageis included between the cryogenic base and a refrigeration source toprovide conductive cooling. A robotic loading and transfer systemaccepts one or more cartridges and loads the cartridges onto thecryogenic base, and a microscopy system images specimens in thecartridges.

In one example, this microscopy system comprises an x-ray source forgenerating an x-ray beam that irradiates the cartridges on the cryogenicbase and a detector for detecting the x-ray beam from the cartridges.

In embodiments, the positioning stage positions the cryogenic base alongthree axes and also rotates the cryogenic base. The warm-cold interfacecomprises a ball and groove configuration for low thermal conductivity.The flexible thermal linkage includes one or more metal wires.

In general, according to another aspect, the invention features, acryogenic x-ray imaging method, comprising generating an x-ray beam thatirradiates specimens, detecting the x-ray beam from the specimens,holding the specimens on a cryogenic base in the x-ray beam, positioningthe specimens in the beam by moving the cryogenic base, and cooling thecryogenic base via a flexible thermal linkage between the cryogenic baseand a refrigeration source.

The above and other features of the invention including various noveldetails of construction and combinations of parts, and other advantages,will now be more particularly described with reference to theaccompanying drawings and pointed out in the claims. It will beunderstood that the particular method and device embodying the inventionare shown by way of illustration and not as a limitation of theinvention. The principles and features of this invention may be employedin various and numerous embodiments without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, reference characters refer to the sameparts throughout the different views. The drawings are not necessarilyto scale; emphasis has instead been placed upon illustrating theprinciples of the invention. Of the drawings:

FIG. 1 is a schematic diagram of a cryogenic x-ray imaging systemaccording to the present invention;

FIG. 2 is a schematic diagram of a kinematic base 150 and cryogenicshield according to the present invention; and

FIG. 3 is a scale drawing of a warm-cold interface of the kinematic baseaccording to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The radiation biology literature uses the International System (SI) unitof the Gray (equal to one Joule of absorbed energy per kilogram of mass)as its unit of radiation dose. At 100 keV in transmission electronmicroscopes (TEM), an electron exposure of 1 e⁻/nm² corresponds to aradiation dose of about 3×10⁴ Gray. From both protein crystallographyand electron microscopy or crystallography data, diffraction spotscorresponding to atom resolution information begin to fade at radiationdoses in the 10⁷-10⁸ Gray range, with diffraction spots corresponding to2-10 nm structural information fading at 10⁸-10⁹ Gray.

In electron microscopy, radiation doses of about 1000 e⁻/nm² or about3×10⁷ Gray lead to the onset of “bubbling” in the specimen, where wateris broken down into OH⁻ and H⁺ and the hydrogen gas will form voids inthe ice matrix when it is unable to diffuse through the ice; enhanceddiffusion may explain the observation that in some cases liquid nitrogentemperatures are preferred to liquid helium temperatures.

In cryogenic x-ray microscopy, excellent structural preservation hasbeen observed at radiation doses as high as 10¹⁰ Gray, without“bubbling”. The absence of “bubbling” is presumably due to somecombination of the lower dose rate relative to cryogenic electronmicroscopy (giving more time for diffusive release of H⁺ through the icematrix) and the lower ratio of absorption in water versus organicmaterials at the 520 eV “water window” photon energies used in theseexperiments. These energies are just below the energy of the oxygenabsorption edge. Sensitive coherence-based “speckle” measurements haveshown that there is no measurable shrinkage of frozen hydrated cryospecimens, at least at doses up to 10¹⁰ Gray.

These studies emphasize the essential nature of cryogenic approaches forx-ray microscopy of hydrated organic specimens such as cells andtissues. For tomography, the specimen must remain constant asprojections from different viewing angles are acquired so that all theindividual views provide true representations of the object that is tobe reconstructed. For spectrum imaging/spectromicroscopy imagesequences, the specimen must not shrink or otherwise change itsmorphology so that all images can be registered to each other to yield aspectrum per pixel for subsequent analysis. For trace element mapping,it is important to not lose side groups that might be bound to the veryelements one is hoping to measure and quantify. Cryogenics is essentialto realize these important x-ray microscopy techniques.

FIG. 1 shows a cyrogenic x-ray imaging system 100 that has beenconstructed according to the principles of the present invention.

In more detail, the system has an x-ray source 110 that generates anx-ray beam 112. In the one embodiment, the source 110 is a beamline of asynchrotron x-ray generation facility. In other embodiments, smallersources are used, such as laboratory sources. For example, laboratorysources that generate x-rays by bombarding a solid target anode withenergetic electrons are one possible source that could be used,including microfocus and rotating anode type sources.

In still other embodiments, the imaging modality is other than x-rays.In one such embodiment, the source generates an electron beam or anoptical beam.

The condenser 114 collects and focuses the x-ray beam 112 from thesource 110. For the full field imaging setup, a suitable illumination ofthe sample 10 is required. This is most conveniently achieved by the useof a zone plate condenser, capillary, or Wolter optic.

When the imaging modality is an electron beam or an optical beam, othercondenser systems are used such as focusing magnets orrefractive/reflective optics, respectively.

A chamber or housing 116 is used to create a controlled environment forthe specimens. The x-ray beam 112 enters the housing 116 through ahousing input window 118. In some examples, the inside of the housing116 is cooled to cryogenic temperatures such as less than 274 Kelvin (K)and usually about 77K, the temperature of liquid nitrogen, or colder. Itis therefore insulated from the surrounding atmosphere. In otherexamples, the housing 116 is capable of holding a vacuum. In such cases,a vacuum pump system 144, such as a system including a turbomolecularpump, is in communication with the housing 116 via a pipe 152 in orderto pull a vacuum within the housing 116.

The x-ray or other beam 112 is projected onto the specimen that iscontained within a cartridge 10. The cartridge 10 is held on ahorizontally extending base 150. This base 150 is a kinematic unit thatpositions the sample cartridge 10 along both the x, y, and z. axes. Thekinematic base 150 further has the capability of rotating thespecimen/cartridge 10 around the y axis to enable the acquisition oftomographic projections at different angles to the axis 115 of the x-raybeam 112.

The kinematic base 150 is held on a mounting plate 122. Then, on top ofthe kinematic base 150 and mounting plate 122, a cryogenic shield 140surrounds the cartridge 10. This cryogenic shield 140 includes a shieldinput beam port 142 through which the beam 112 passes to the samplecartridge 10. A shield output beam port 144 of the cryogenic shield 140allows the beam to exit after passing through the specimen/cartridge 10.

A refrigeration source 124 is preferably located within the housing 116.It is connected via a heat transfer element 125 to the cryogenic shield140. In one example, this refrigeration source 124 is a refrigerationunit. In other examples, the refrigeration source 124 is a dewar orother container containing liquid nitrogen. The heat transfer element125 is constructed from a high thermal transfer material such as braidedcopper cable.

The beam 112 from the sample cartridge 10 exits the cryogenic housing116 through a housing output port 126. An x-ray objective 128 collectsx-rays 112 from the specimen and images the x-ray beam 112 onto adetector system 130. In a current embodiment, the objective 128 is aFresnel zone plate.

In examples where the beam 112 is an optical beam, the image is formedwith refractive or reflective optics.

The detector system 130 is preferably a high-resolution, high-efficiencyscintillator-coupled CCD (charge coupled device) camera system fordetecting x-rays from the specimen. In one example, a camera system 130as described in U.S. Pat. No. 7,057,187, which is incorporated herein bythis reference in its entirety, is used.

A robotic loading and unloading system is provided in the preferredembodiment. Microscopy specimens are delicate and have a poor chance ofsurviving repeated handling. For this reason it is good practice tomount them once in a cartridge, and then handle that cartridge insubsequent operations. Cartridges 10 are loaded into the system 100 on ashuttle 176. A robot system 170 then individually loads and unloads thecartridges 10 onto kinematic base 150.

Cartridge covers are preferably used to prevent contamination buildup onthe specimen during the various handling steps. Further the cartridges10 preferably all share a common design in the top that is grabbed bythe robot system's gripper 174 and for the end that is inserted into thekinematic base 150. Preferably, a unified base can support a variety ofspecimen mounting schemes. For example, one type of cartridge might useclamping rings for standard 3 mm TEM grids, another might use amicro-fabricated silicon stalk to minimize x-ray fluorescence backgroundwhile maintaining good dimensional stability and thermal conductivity,while yet another might use a thin-walled capillary for the mounting oftomography specimens.

A horizontal linear travel stage 175 is used to move the shuttle base176 from a position well out of the way of the kinematic base 150, to aseries of locations that put each of the cartridges 10 or cartridgesslots in the shuttle 176 directly above the center of the kinematic base150 and the loading port 146 formed in the cryo shield 140. A robot arm172 the picks the cartridges with the gripper 174 and transfers thecartridges 10 between the kinematic base 150 and the cartridge slots ofthe shuttle 176, accessing the kinematic base 150 via the loading port146.

The robot system 170 preferably has a vertical linear travel stage 172upon which the gripper 174 is mounted. A fiberglass insert providesthermal isolation for the gripper end 174 which is in turn conductivelycooled using a copper braid to a dewar, in one example. This requiresaccess to the specimen from above, and either enough “headroom” in thechamber 116 to hold the vertical linear travel stage upon which therobot grabber is mounted, or a port with a linear feedthrough.

FIG. 2 shows the details of the kinematic base 150 and shield 140.

A small, low-mass cryo base 152 is mounted on a high-temperaturerotation and/or nanopositioning stage 154 that is supported on themounting plate 122. The nanopositioning stage 154 positions and movesthe cryo base 152 and thus the specimen in the cartridge 10 along the x,y and z axes to position the region of interest of the specimen withinthe x-ray beam 115, and also preferably rotates the specimen about the yaxis.

A warm-cold interface 156 separates the nanopositioning stage 154 fromthe cryo base 152. It is constructed from an interface material and hasa geometry with low mechanical creep and low thermal conductance.

The cold cryo base 152 mainly “sees” the area of the cryo shield 140,radiative heat transfer into the specimen 10 is thus greatly reduced.The dominant heat transfer path becomes that of the warm-cold interface156, which has both high mechanical stiffness and low thermalconductivity. In this way only modest cooling power (well below 100milliWatts (mW)) must be supplied to the cryo base 152. This ispreferably supplied by “weak” heat conductors 158 which involve very lowmechanical coupling force between the cryo base 152 and the cryo shield140 for rotations up to ±90 of the cryo base by the movement of thenanopositioning stage 154 or translations of several millimeters.(Initial cool-down involves moving a raised surface on the cryo baseinto strong contact with a cold “finger” from the cryo shield). Thiscryo base 152 is normally kept cold in the microscope 100 at all times.

Key thermal design considerations for this approach include thefollowing: Gas conductivity becomes negligible at pressures of belowabout 10⁻⁴ torr, and pressures well below this are needed to minimizeice buildup on cryo specimens. Because the thermal conductivity ofhigh-purity copper increases at lower temperatures, weak heat conductors158 comprise a number of copper wires that can provide good thermalcooling power, such as less than 500 wires. As an example, 150 wires inparallel, each 100 micrometers (μm) in diameter and 50 millimeters (mm)long, can provide 120 mW of cooling power over a temperature differenceof 10 K between the cryo shield 140 that is cooled by the heatconduction through heat transfer element 125 to the refrigeration source124. The wire conductors 158 are much longer than the distance (D)between the outer wall of the base 152 and the shield 140 so that thebase 152 is moved and rotated freely by the nanopositioning stage 154.In one example, the length of the wire conductors 158 are more than 5times distance D.

FIG. 3 shows an embodiment of the warm-cold interface 156.

In more detail, the warm-cold interface 156 comprises an upper member312 on which the cryogenic base 152 is placed and lower member 310 thatis secured to the nanopositioning stage 154.

Both the upper member 312 and lower member 310 have low emissivitycoatings, especially on the two surfaces that face each other. Forexample, using a conservative estimate for the emissivity (ε) of highlypolished gold of ε=0.05 (as opposed to the ε=0.018-0.035 values given inpublished tables), the radiative heat transfer between two 25 mm diskswhen one is at 100 K and the other at 300 K is only about 11 mW. Thermalconductivity then becomes the dominant path. This is controlled by usinga ball-on-flat mounting approach and with both low conductivitymaterials. Preferably, both the upper member 312 and lower member 310are constructed from fused silica or an infrared glass. AMTIR-1, fromAmorphous Materials Inc, for example, has 5× lower conductivity andnearly equal stiffness.

The current ball-on flat approach to thermal isolation uses threerecesses 316A, 316B, 316C are formed in the lower member 310. In thepreferred embodiment, each of these three recesses 316A, 316B, 316Ccomprises three planar surfaces in the general form of a pyramid. In analternative design, the three recesses 316A, 316B, 316C are in the formof a cone. Low thermal conductivity balls or spheres 314A, 314B, 314Care each placed in a corresponding one of the three recesses 316A, 316B,316C. The upper member 312 has a corresponding, mirrored series ofrecesses that receive the balls 314A, 314B, 314C. This creates rigid yetlow thermal conductivity interface.

Depending on the choice of materials and the force applied, a thermalconduction power of no more than 20-50 mW can be obtained between thecryo base 152 and the warm nanopositioning stage 154. This ball-on-flatsystem implemented in the upper member, 312, lower member 310 and ballsor spheres 314A, 314B, 314C also has the advantage of being naturallysuited to a kinematic mounting system, where no mechanical stress isinduced that would otherwise lead to mechanical drift. Other designconfigurations can include the commonly used ball-groove-flat kinematicmounting system.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A cryogenic imaging system, comprising: akinematic base that receives cartridges on a cryogenic base, with eachcartridge carrying a specimen and further includes a positioning stageand a warm-cold interface between the positioning stage and thecryogenic base; a flexible thermal linkage between the cryogenic baseand a refrigeration source to provide conductive cooling; a roboticloading and transfer system that is able to accept one or morecartridges and load the cartridges onto the cryogenic base; a source forgenerating an x-ray beam that irradiates the cartridges on the cryogenicbase; and a detector for detecting the x-ray beam from the cartridges.2. A system as claimed in claim 1, wherein the positioning stagepositions the cryogenic base along three axes.
 3. A system as claimed inclaim 1, wherein the positioning stage rotates the cryogenic base.
 4. Asystem as claimed in claim 1, wherein the warm-cold interface comprisesa ball and groove configuration.
 5. A system as claimed in claim 1,wherein the warm-cold interface comprises a ball and cone configuration.6. A system as claimed in claim 1, wherein the flexible thermal linkagecomprises one or more metal wires.
 7. A system as claimed in claim 1,further comprising cryogenic shield surrounding the cryogenic base, thecryogenic shield being cooled by the refrigeration source.
 8. A systemas claimed in claim 7, wherein the flexible thermal linkage extendsbetween the cryogenic base and the cryogenic shield.
 9. A system asclaimed in claim 7, wherein the cryogenic shield comprises beam portsthrough which the x-ray beam passes.
 10. A system as claimed in claim 7,wherein the cryogenic shield comprises loading ports through which therobotic loading and transfer system transfers the cartridges.
 11. Asystem as claimed in claim 1, wherein the robotic loading and transfersystem comprises a shuttle holding multiple cartridges and a gripper fortransferring cartridges between the shuttle and the cryogenic base.